CD4+ regulatory T (Treg) cells play a critical role in autoimmune diseases by suppressing the CD4+ and CD8+ effector cell responses, but inhibit immune responses needed for effective cancer immunotherapy. We recently demonstrated the presence of antigen-specific CD4+ Treg cells in melanoma and prostate tumor-infiltrating T cells. These observations may explain, at least in part, why attempts to elicit strong and durable antitumor responses with cancer vaccines (comprising MHC class l-restricted tumor antigens or peptides) have been largely unsuccessful. The goals of this project are to investigate whether the presence of Treg cells in prostate cancer cause immune suppression and tolerance, and whether antitumor immunity can be enhanced by reversing the suppressive function of Treg cells. Research in the applicant's laboratory has established over 50 prostate tumor infiltrating T cells (PTILs). Both CD4+ and CD8+ Treg cells from several PTILs showed antigen specificity and potent suppressive activity. More importantly, we recently demonstrated that the suppressive function melanoma-derived Treg cells could be specifically reversed by TLR ligands. These preliminary studies prompted us to test our hypothesis that the suppressive function of prostate cancer-derived CD4+ and CD8+ Treg cells could be regulated or reversed by cytokines and TLR signaling. These unique prostate Treg cells lines, together with several newly developed technologies, should enable identification of important ligands for CD4+ and CD8+ Treg cells, permitting in turn more rigorous testing of a novel concept - that reversing the suppressive function of CD4+ and/or CD8+ Treg cells may boost the effectiveness of cancer immunotherapy. To test our novel concept and hypothesis, we propose three specific research aims: (1) characterize different subsets of PTILs on the basis of phenotypic markers, cytokine profiles and suppressive mechanisms;(2) use established CD4+ Treg cell lines/clones to identify genes encoding the ligands of these cells and then characterize their roles in regulating the activation of Treg cells;(3) dissect the immunosuppressive mechanisms and regulation of Treg cells by TLR signaling to gain critical information needed to test our hypothesis that reversing the suppressive function of CD4+ Treg cells would enhance antitumor immunity in vivo. The strategies emerging from this 5-year proposal will be applied to the regulation of Treg cell function in different human tumors to verify that a shift in the CD4+ Treg/effector cell balance through TLR signaling or cytokines is indeed conducive to more effective cancer immunotherapy. A positive outcome of these studies would open new opportunities for treating cancer patients and perhaps infectious and autoimmune diseases as well.